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Abstract

The present authors have previously reported a novel approach to genetically engineer Salmonella typhimurium for the medically important therapeutic strategy of using bacterial agents to target malignant tumors in a breast cancer tumor‑bearing nude mouse model. However, studying an immunocompromised mouse model for cancer therapy is insufficient, as certain crucial information about the influence of the immune system may be missing. In the present study, inoculation of the Salmonella strain, YB1, into a colon cancer tumor‑bearing immunocompetent mouse model was investigated. The present study determined the tumor targeting efficiency, antitumor potential, the effects of multiple treatments and the systemic toxicity. Intravenous inoculation of YB1 in BALB/c mice exhibited high antitumor effects and also greatly increased the tumor targeting ability and safety compared with the previously‑reported nude mouse model. In addition, repeated administration of YB1 further enhanced this effect. Furthermore, no marked toxicity was observed with YB1 treatment, while the VNP20009 and SL7207 strains demonstrated certain adverse effects. The findings of the present study indicate that the YB1 strain is effective and safe in targeting a colon cancer tumor in an immunocompetent mouse model.

Introduction

The potential use of certain obligate and
facultative anaerobic bacteria in cancer treatment has been
investigated for numerous years (1).
Bacteria, including Bifidobacterium, Clostridium and
Salmonella, have been demonstrated to preferentially target
and replicate in the hypoxic and necrotic regions of tumors,
resulting in tumor repression (2–9). In our
previous study, in order to strengthen the tumor hypoxia targeting
efficiency and antitumor effect, a synthetic biology approach was
used to generate a novel Salmonella strain, YB1 (10). The principle of YB1 is to regulate an
essential gene (asd) of Salmonella using a
hypoxia-induced promoter (10). Since
Salmonella is sensitive to the expression of the asd
gene, any leaky expression will destroy the entire regulation.
Therefore, a genetic circuit was designed to neutralize the
leakage, which was controlled by an anaerobic promoter in order to
regulate forward transcription of asd, and another aerobic
promoter was constructed to generate anti-sense asd mRNA
(10). With this combination, YB1
only survived in an atmosphere with <0.5% O2, which
is equivalent to the anaerobic conditions inside solid tumor tissue
(11), and the growth of YB1 was
inhibited when exposed to increased oxygen levels that are
equivalent to those in a normal organ (2–9% O2) without
additional diaminopimelic acid (DAP) (10,12). In
the breast tumor-bearing nude mouse model, YB1 specifically
colonized and proliferated in hypoxic/necrotic areas of the tumor,
and retarded tumor growth (10).

In the present study, the antitumor effects of the
YB1 strain were further investigated. The tumor targeting
efficiency and safety evaluation were investigated in a solid tumor
model using the colon cancer cell line, CT26. In addition,
inoculation of YB1 in an immunocompetent mouse model (BALB/c mice)
was compared with the previously-reported immunocompromised mouse
model (nude mice). Furthermore, multiple YB1 injection treatments
were assessed and the treatment of different tumor sizes was
investigated. The present study, using the genetically engineered
YB1 strain, provides preclinical data on the efficiency and safety
of Salmonella mediated cancer therapy, which may further
facilitate its optimization to improve the anticancer effect and
reduce adverse effects in future clinical trials.

Materials and methods

Bacteria, cell lines and animals

The Salmonella typhimurium strain, SL7207,
was obtained from lab stock (13).
The VNP20009 strain was purchased from the American Type Culture
Collection (#202165; Manassas, VA, USA). The YB1 strain was
constructed as described previously (10). Bacteria were cultured in lysogeny
broth (LB) medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented
with chloramphenicol and DAP (Sigma-Aldrich) at 37°C. The CT26
colon carcinoma cell line was provided by Dr Songyue Zheng
(University of Hong Kong, Hong Kong, China). Cells were cultured in
high-glucose Dulbecco's modified Eagle's medium containing 10%
fetal bovine serum and 1% penicillin-streptomycin (Gibco Life
Technologies, Carlsbad, CA, USA). Four-week-old female BALB/c nude
mice (weight range, 16–20 g) and eight-week-old BALB/c mice (weight
range, 18–20 g) were purchased from the Laboratory Animal Unit of
The University of Hong Kong. The research protocols were approved
and followed by the committee on the Use of Live Animals in
Teaching and Research of the University of Hong Kong (CULATR no.
2689-12). Antibiotic and chemical working solutions were prepared
as follows: Chloramphenicol, 25 µg/ml in methanol; DAP, 50 µg/ml in
water.

Tumor-bearing mouse model

A total 5×105 CT26 cells were injected
into the fat pad of the chest in nude mice or BALB/c mice. The
tumor volumes were calculated using the following formula: 4/3 π
(height × width2)/8. When the tumors grew to ~500 or 100
mm3, the mice underwent bacterial treatment and were
grouped as follows: YB1-treated BALB/c group; YB1-1-treated nude
group; VNP20009-treated BALB/c group; and SL7207-treated BALB/c
group. Phosphate-buffered saline (PBS) treatment was used as a
control. If the tumor size reached 20 mm in height or the volume
was ≥4,000 mm3, the mouse was euthanized (14). To measure YB1 distribution following
treatment at different time points in nude mice and BALB/c mice,
5×107 colony-forming units (CFU) of YB1, VNP20009 or
SL7207 were injected intravenously into CT26 tumor-bearing mice
through the tail vein. The mice were sacrificed by intraperitoneal
injection with pentobarbitone (University of Hong Kong) at
different time points (day 1, 3, 7 or 11). The calculation of YB1
in tissues, survival rates and body weight analysis followed the
methods previously described (10).

Tumor targeting potential and
clearance of YB1

The tumor targeting potential and the clearance of
YB1 in normal tissues were compared in immunocompetent (nude) and
immunocompromised (BALB/c) mice. CT26 tumor cells were injected
subcutaneously into nude and BALB/c mice. When the tumor volumes
reached ~500 mm3, a single shot of YB1 was injected
intravenously through the tail vein. At the indicated time points
(day 1, 3, 7 or 11), the mice were euthanized by intraperitoneal
injection with pentobarbitone and the liver, spleen and tumor were
isolated, homogenized in LB and cultured on LB agar plates
supplemented with antibiotics and DAP.

Multiple-shot YB1 treatment in BALB/c
mice CT26 cancer model

A total of 1×105 CT26 tumor cells were
injected subcutaneously into 10 BALB/c mice. When the tumor volumes
reached ~500 mm3, mice in the ‘single-shot YB1’ group
(n=5) were intravenously injected with a single-shot of YB1,
whereas mice in the ‘multiple-shot YB1’ group (n=5) received an
intravenous injection of YB1 every two days (days 0, 2, 4 and 6).
The tumor sizes in the two groups were measured on days 0, 1, 3, 6,
8 and 10. On day 10, all mice were sacrificed by intraperitoneal
injection with pentobarbitone. The liver, spleen and tumor were
then isolated, homogenized in LB and cultured on LB agar plates
supplemented with DAP to determine YB1 distribution.

Single-shot of different Salmonella
strains in small CT26 tumor models

A total of 1×105 CT26 tumor cells were
injected subcutaneously into 20 BALB/c and 10 nude mice. When the
tumor volumes reached ~100 mm3, a single-shot of YB1
(n=5), VNP20009 (n=5), SL7207 (n=5), or PBS (n=5) was injected
intravenously into BALB/c mice, whereas nude mice were
intravenously injected with a single-shot of YB1 (n=5) or PBS
(n=5). The tumor sizes in all groups were measured on days 0, 1, 3,
6, 8, 10 and 12. On day 12, all mice were sacrificed by
intraperitoneal injection with pentobarbitone. The liver, spleen
and tumor were then isolated, homogenized in LB and cultured on LB
agar plates supplemented with DAP to determine the distribution of
YB1, VNP20009 and SL7207.

Statistical analysis

Student's t-test was used to analyze the
significance of the results of the CFU test and tumor growth
measurements. Analyses were performed using GraphPad Prism 5
(GraphPad Software, Inc., La Jolla, CA, USA). Data are expressed as
the mean ± standard deviation and P<0.01 was considered to
indicate a statistically significant difference.

Results

Accumulation of YB1 in CT26 tumors and
normal tissues in nude and BALB/c mice

For the nude mice group, the results were similar to
those observed in our previous study in a breast cancer
tumor-bearing nude mouse model (10).
Between 1×105 and 1×106 CFU/g of bacteria
were observed in all the tested tissues on day 1 (Fig. 1A). After one day, the YB1 levels in
the liver and spleen declined rapidly. In the tumor tissues, the
YB1 levels increased until they reached a plateau of
1×108 CFU/g at day 3, which remained stable until day 11
(Fig. 1B–D). The tumor to liver ratio
was 9,000:1 on day 3 (Fig. 1B;
P<0.05) and 300,000:1 on day 7 (Fig.
1C; P<0.05). By day 11, YB1 was completely eliminated from
the liver and spleen (Fig. 1D,
P<0.01). Notably, the YB1 levels reduced much more quickly in
the BALB/c mice compared with the nude mice. Within 24 h after
treatment with YB1, the YB1 levels reduced to ~1×102
CFU/g in the liver and spleen (Fig.
1A), while between days 3 and 11 no YB1 was detected in the
normal tissues (Fig. 1B–D). YB1
levels in the tumor demonstrated a similar pattern in nude mice,
which reached a plateau of 2–4×108 CFU/g at day 3 that
was maintained until day 11 (Fig.
1B–D).

Since a large amount of YB1 accumulated in the
tumor, its antitumor effect was measured. Tumor growth (from a
volume of ~500 mm3 at the time of bacterial inoculation)
following a single-shot YB1 treatment in BALB/c mice was initially
inhibited for 3 days and then delayed compared with the PBS-treated
group from days 3–10 (P<0.01 on days 3 and 8; P<0.001 on days
6 and 10) (Fig. 2A). Since YB1 was
cleared in normal tissues within one day (Fig. 1), YB1 treatment was increased in
tumor-bearing BALB/c mice via intravenous injections every two days
(on days 2, 4, 6 and 8). The tumor growth was significantly
repressed (P<0.001 on days 8 and 10) and the volume of the
tumors remained ~500 mm3 in the 10 subsequent days
(Fig. 2A). On day 10 following YB1
treatment, the single-shot and multiple-shot groups of mice were
euthanized by intraperitoneal injection with pentobarbitone, and
the bacterial number in different organs was counted. The residual
YB1 in the liver and spleen of the two groups was eliminated. In
the tumor tissues, the YB1 levels in the multiple-shot group were
increased compared with the single-shot group (P<0.05; Fig. 2B).

Antitumor effect with single-shot of
different Salmonella strains in small CT26 tumor models (~100
mm3)

To determine the antitumor ability of YB1 in small
tumors (~100 mm3), a single-shot of YB1 was
administrated to CT26 tumor-bearing nude and BALB/c mice (Fig. 3A and B). The results demonstrated that
YB1 accumulated at approximately the same level in the two mouse
models (Fig. 3C). The growth of the
small tumors following inoculation with YB1 varied markedly, and
tumor growth was delayed in nude mice but not in BALB/c mice
(Fig. 3A). However, the overall
growth of the tumor in BALB/c mice treated with a single-shot YB1
was significantly reduced compared with the PBS-treated mice,
within the 12-day observation period (Fig. 3B).

The VNP20009 treatment repressed tumor growth in
BALB/c mice for 6 days; however the tumor continued to grow after
day 6 (Fig. 3B). The SL7207-treated
mice demonstrated approximately the same pattern in tumor growth
repression as YB1 inoculation in BALB/c mice (Fig. 3B). However, although YB1, VNP20009 and
SL7207 accumulated at similar levels in the tumor (2×108
CFU/g), significant differences were observed in the normal tissues
(Fig. 3C). SL7207-inoculated mice
demonstrated an uncontrolled infection as the bacterial level in
the liver and spleen increased to 1×108 CFU/g (Fig. 3C). In VNP20009-treated mice, the
bacterial levels were slightly reduced compared with the mice
treated with SL7207, but were still ~1×107 CFU/g in the
liver and spleen (Fig. 3C).

Evaluation of the adverse effects of
bacterial infection on weight gain and survival rate

Body weight and survival rate are major evaluation
makers of treatment toxicity analysis for preclinical studies
(15). Therefore, the adverse effects
of the bacterial infection strategies used in the present study
were assessed based on the survival rate and body weight. Mice with
small CT26 tumor models (~100 mm3) that were treated
with single-shot YB1, VNP20009 or PBS survived during the 12-day
observation period. However, a number of SL7207-treated mice
succumbed on day 8, and the overall survival rate within the 12-day
observation period was 40% (Fig. 4A).
Following the first day of inoculation, all the treatment groups
demonstrated significant body weight loss compared with the
PBS-treated group (YB1, P<0.01; VNP20009 and SL7207, P<0.05);
however, only the YB1-treated group started to recover after day 1
(P<0.01), whereas the other groups demonstrated continuous
weight loss throughout the 12 days of observation (VNP20009,
SL7207, P<0.001 on days 6–12). Although no mice succumbed in the
VNP20009-treated group, their health condition was poorer than the
YB1 group, due to the reduction in body weight following treatment
(Fig. 4B).

Discussion

The physiology of solid tumors is different compared
with normal tissues in certain aspects (16–20).
Compared with normal organs, solid tumors consume a larger amount
of oxygen and nutrients, a greater number of blood vessels supply
the tissue and the vasculature within the tumor is highly abnormal
with leaky vessel walls and reduced flow (16–18).
Although tumors continuously generate new blood vessels, large
regions of hypoxia are frequently observed in solid tumors
(11,21,22). These
regions of hypoxia lead to problems in cancer treatment and can
result in resistance to radiotherapy or chemotherapy (21). However, the microenvironment of the
solid tumor provides a haven for certain obligate or facultative
anaerobic bacteria, such as Salmonella (23,24). When
attenuated Salmonella strains are used as an anticancer
vector, they are preferentially colonized in tumors with the ratio
of titer being 1,000–10,000:1 in tumor over normal tissues. They
may also evade monitoring by the immune system, and thus replicate
and accumulate in the hypoxia region for long periods of time
(1,7,8,25). Although Salmonella
preferentially accumulates in tumor tissues, a proportion colonizes
the normal tissues (10,26). In our previous study, recombineering
technology was used to convert a Salmonella typhimurium
strain into an ‘obligate’ anaerobe without otherwise interfering
with the function of the bacterium. This avoids the problem of
infection in normal aerobic tissues, as the modified strain lyses
under these conditions. When in the hypoxic regions of a tumor, the
bacterium thrives and functions as the wild type form as it is not
compromised by an attenuation process. The efficacy of this
approach was demonstrated by the effective tumor targeting and
regression without damage to the normal organs in a situation where
the unmodified parent strain is lethal to the host (10).

In our previous study (10), the animal model used to investigate
Salmonella treatment included nude mice, which are deficient
in T cell function. Mutations in the Foxn1 gene in nude mice
results in thymic aplasia and a lack of T cells, although the B
cells remain unchanged (27). The
immune system may aid bacteria to prevent cancer progression
(28). A major obstacle for cancer
immunotherapy is the ability of tumors to generate a
microenvironment to evade the monitoring of the immune system.
Although numerous methods have been developed to induce cancer
immunity and certain interventions have demonstrated remarkably
potent antitumor specific cytotoxic responses (29–32), the
majority of tumors did not regress following these treatments and
continued to grow even when tumor specific T cells were introduced
in the circulation (29,33). The reason for this may be due to the
fact that their entry into the tumor masses was limited, or their
functions were weakened and defected due to the downregulation of
the specific target of antigen or major histocompatibility complex
molecules inside the tumor (34).
However, it has been demonstrated that, when combined with
bacterial infection, the ability of the immune system to kill tumor
cells was restored, which may aid in the goal to overcome immune
escape mechanisms (28). Combined
with Salmonella infection, tumor cells were destroyed by the
immune system. In addition, it was demonstrated that tissue debris
was captured and taken up by endogenous antigen-presenting cells,
and presented on the cell surface of naïve T cells in order to
generate tumor-specific T cells (35). Therefore, investigating the modified
Salmonella strain in immunocompetent mice is an important
experiment in moving towards clinical trials.

In the present study, an immunocompetent mouse model
(BALB/c mice) was used, in which the clearance speed of YB1 in
normal tissues was much faster compared with that in nude mice.
While nude mice required at least 11 days to completely eliminate
YB1, BALB/c required 1–2 days (Fig.
1). This may be due to a more efficient immune system in BALB/c
mice compared with nude mice. In light of this improvement, the
mice were treated with repeated injections. With high frequency
boosting of YB1 every two days for a total of five treatments, the
tumor growth was significantly repressed. These results indicated
that the involvement of T cells did enhance the antitumor effects
of YB1 treatment. Notably, when BALB/c mice with relatively small
tumors (~100 mm3) were treated, a single injection was
sufficient to prevent tumor progression (Fig. 3). Using the genetically engineered YB1
strain of Salmonella in cancer therapy demonstrated a
promising antitumor effect, as well as high safety (Fig. 4). In conclusion, the approach of
engineering Salmonella offers a feasible, effective and safe
option for delivering bacteria in cancer therapy. In future
studies, live animal imaging (intravital) technology may be applied
to investigate the process of bacteria targeting tumors in more
detail.

Acknowledgements

The present study was supported by a grant by the
National Science Foundation of China (no. 31200639), awarded to Dr
Bin Yu and Dr Lei Shi.